Method of purifying virus-like particles

ABSTRACT

A method of producing purified FMDV VLPs, comprising contacting cells containing FMDV VLPs with a lysis buffer and allowing the cells to lyse, the lysis buffer comprising 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl 2 , and 1% Triton X-100, wherein the lysis buffer does not contain EDTA; centrifuging a solution; and removing a supernatant from the solution, the supernatant containing the purified FMDV VLPs.

CROSS REFERENCE TO RELATED APPLICATION

This U.S. Non-provisional application claims the benefit of priority toU.S. application Ser. No. 17/326,023 filed on May 20, 2021 which claimspriority to Provisional Application No. 63/028,694 filed on May 22,2020, the disclosures of all of which are herein incorporated byreference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support awarded by the U.S.Department of Homeland Security. The United States Government hascertain rights in this invention.

TECHNICAL FIELD

The present disclosure relates to a method of purifyingvirus-like-particles (VLPs), such as VLPs from foot-and-mouth diseasevirus (FMDV). The method produces an increased amount of purified VLPs,and at a lower cost, than that obtained by previous methods. The presentdisclosure also relates to a method of inoculating an animal with thepurified VLPs to induce an immune response to FMDV, and to formulationsof the purified VLPs such as in a vaccine.

BACKGROUND

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. This background isnot expressly or impliedly admitted as prior art against the presentdisclosure.

The foot-and-mouth disease virus (FMDV) is the causative agent of ahighly infectious and sometimes fatal disease that affects cloven-hoofedanimals such as cattle, pigs, sheep, goats, and deer. There are sevenmajor antigenically distinct FMDV serotypes (A, O, C, Asia 1, SAT 1, SAT2, and SAT 3), and each serotype contains multiple subtypes ortopotypes. Currently, serotype O is the most common serotype worldwide.Example FMDV strains include A24 Cruzeiro, A2006 Turkey, O1 Campos, O1Manisa, O Pan Asia II, O Hong Kong, C3 Indaial, Asia 1 Shamir, SAT1KNP91, SAT3 ZIM91, and SAT2 Egypt 2010.

Foot-and-mouth disease outbreaks cause significant agro-economic losseswith severe implications for animal farming throughout the world. FMDVcan be spread to uninfected livestock by direct contact, throughaerosols from infected domestic or wild animals, or throughcontamination of feed and/or equipment. Containment of an FMDV outbreakdemands considerable effort and expenses for vaccination, vigilant andstrict monitoring of livestock, and culling and disposal of infectedlivestock.

Commonly used foot-and-mouth disease vaccines utilize whole virus thathas been chemically inactivated. This vaccine platform has severallimitations and shortcomings. Animals immunized with inactivated virusare difficult to distinguish from naturally infected animals. Theefficacy of the vaccine formulations is limited by immunogenicinstability and short vaccine shelf life that results in a loss ofpotency upon transportation or storage and subsequent induction ofinsufficient immunity or immunity of a short duration. Furthermore, theset-up and running costs of producing the FMDV vaccine in potent formand securing and maintaining its production facilities are very high.

Newer generations of vaccines utilize FMDV polypeptides expressed fromnon-infectious vectors, such as a plasmid, to produce immunogenic FMDVantigens. These platforms utilize expression of the FMDV 3C protease toprocess and cleave the expressed FMDV P1 precursor polypeptide into thevirus structural proteins VP0, VP3, and VP4. The fusion protein VP0 is acombination of the structural proteins VP4 and VP2, the processing ofVP0 into VP4 and VP2 is not performed by the 3C protease and occurs byan unknown mechanism during encapsulation. The fully processedstructural proteins VP4, VP2, VP3, and VP1, can formvirus-like-particles (VLPs) for use in vaccines, VP0 can also beincorporated into VLPs in place of VP4 and VP2. VLPs structurallyresemble viruses but are non-infectious because they do not contain anyviral genetic material. For the purpose of producing a vaccine,extracted antigen is considered a VLP if it elicits a protective immuneresponse against virus challenge in the target species. Additionaldetails of technology related to these VLP vaccines are described inU.S. Pat. Nos. 9,975,926; 10,513,542; 10,604,548; 10,385,319; and10,308,927, each of which is incorporated by reference in its entirety.

BRIEF SUMMARY

According to a first aspect, the present disclosure provides a method ofproducing purified VLPs that includes contacting cells containing VLPswith a lysis buffer containing Tris-HCl, NaCl, MgCl₂, and Triton X-100,and allowing the cells to lyse. In various embodiments, the lysis bufferdoes not contain any EDTA. After the cells have lysed, the methodfurther includes centrifuging a solution of the lysed cells and removingthe supernatant from the centrifuged solution. The supernatant containspurified VLPs

According to various embodiments of the method, the VLPs are FMDV VLPs,and the embodiments are directed to a method of producing purified FMDVVLPs.

According to various embodiments of the method of producing purifiedVLPs, the lysis buffer contains 20 mM Tris-HCl, 150-200 mM NaCl, 3 mMMgCl₂, and 1% Triton X-100 (w/v).

According to some embodiments, the method of producing purified VLPsalso includes centrifuging the supernatant through a centrifugal filterwith a 1,000,000 Da cut off.

According to some embodiments, the method of producing purified VLPsalso includes centrifuging the supernatant through a cesium chlorideand/or a sucrose gradient and collecting purified VLPs from thegradient.

In a second aspect, the present disclosure provides a method ofinoculating an animal to induce an immune response against FMDV. Themethod of inoculating includes (i) providing purified FMDV VLPs thathave been produced by a method that includes contacting cells containingFMDV VLPs with a lysis buffer that contains Tris-HCl, NaCl, MgCl₂, andTriton X-100, centrifuging a solution of the lysed cells, and removingthe supernatant containing the purified FMDV VLPs; and (ii) inoculatingthe animal with an effective amount of the purified FMDV VLPs.

In a third aspect, the present disclosure provides a compositioncontaining purified FMDV VLPs produced by a method that includescontacting cells containing FMDV VLPs with a lysis buffer that containsTris-HCl, NaCl, MgCl₂, and Triton X-100; centrifuging a solution of thelysed cells; and removing the supernatant containing the purified FMDVVLPs.

According to various embodiments, the composition containing FMDV VLPsis a vaccine for foot-and-mouth disease containing an effective amountof the purified FMDV VLPs.

BRIEF DESCRIPTION OF THE DRAWINGS

An appreciation of the disclosure and many of the attendant advantagesthereof may be understood by reference to the accompanying drawings.Included in the drawings are the following figures:

FIG. 1A is a plasmid map of the plasmid pJJP O1M-3C(L127P)-GLuc, thatwas used to produce VLPs in transfected cells. FIG. 1B is a Western blotusing F14 antibody for the detection of VP0 and VP2 proteins fromsupernatants extracted with lysis buffers LB1 through LB6.

FIG. 2A shows the results of luciferase detected in relative luciferaseunits per half second of supernatant extracted with lysis buffers LB3,LB7, LB8, LB9, and LB10. FIG. 2B is a western blot using F14 antibodyfor the detection of VP0 and VP2 proteins from supernatants extractedwith lysis buffers LB3, LB7, LB8, LB9, and LB10.

FIG. 3 shows the resulting band patterns observed after size-basedpurification by cesium chloride gradient of VLPs extracted from CHO-K7and HEK293-T cells using lysis buffer LB3 or LB9. Banding and materialbetween the black marks represent antigen and the appropriate size rangefor FMDV VLPs.

FIG. 4A shows the band pattern from a cesium chloride gradientpurification of VLPs extracted from HEK393-T cells using lysis bufferLB9.

FIG. 4B Upper (U) and lower (L) bands were extracted and run on aWestern blot, alongside an aliquot that was not applied to a cesiumchloride gradient (Pre CsCl₂), using the F14 antibody for detection ofFMDV proteins VP0 and VP2.

FIG. 5 shows cesium chloride gradients on samples extracted using lysisbuffer LB9 from HEK293-T cells producing VLPs from different FMDVserotypes and strains. A2006=A2006 Turkey; A24=A24 Cruzeiro; Asia1=Asia1 Shamir; SAT2=SAT2 Egypt 2010; O1C=O1 Campos; OPA2=O Pan Asia II; OHK=OHong Kong.

FIG. 6 shows Western blots using F14, 12FE9, and 6HC4 antibodies onsamples extracted using lysis buffer LB9 from HEK293-T cells transfectedwith plasmids encoding different FMDV serotypes and strains. A24=A24Cruzeiro; A06=A2006 Turkey; A1S=Asia1 Shamir; O1C=O1 Campos; O1M=O1Manisa; OPA=O Pan Asia II; OHK=O Hong Kong; SE2=SAT2 Egypt 2010.

FIG. 7 shows the highest observed VNTs for each pig during the course ofthe challenge studies sorted in relation to VLPs extracted with eitherQLB (black) or LB9 (gray).

FIG. 8 shows The VNTs at the time of challenge of all animals within atreatment group were averaged together and are presented.

DETAILED DESCRIPTION

In addition to its other benefits, the present disclosure provides a newand advantageous method to produce purified VLPs. In a first aspect, themethod utilizes a lysis buffer that results in an increased amount ofpurified VLPs, and at a much lower cost, than that obtained by previousmethods.

For purposes of the present invention, “foot-and-mouth disease virus” orthe acronym FMDV refers to any of the seven major FMDV antigenicallydistinct virus serotypes, i.e. A, O, C, Asia 1 and South AfricanTerritories 1, 2 and 3, as well as the multiple subtypes or topotypesthat exist within each serotype. In some embodiments, the VLPs are fromone or more of FMDV strains A24 Cruzeiro, A2006 Turkey, O1 Campos, O1Manisa, O Pan Asia II, O Hong Kong, C3 Indaial, Asia 1 Shamir, SAT1KNP91, SAT3 ZIM91, and SAT2 Egypt 2010. Infection with any one serotypedoes not confer protective immunity against another.

The FMDV is a non-enveloped picornavirus (belonging to the genusAphthovirus of the family Picornaviridae) with a single-stranded genomicRNA of between 7,500 to 8,000 nucleotides. The capsid, which is theprotein shell of the virus, is made up of 60 copies of each of the fourstructural proteins VP1, VP2, VP3 and VP4. The precursor protein VP0, afusion of VP2 and VP4, can also be incorporated into the capsid. Inembodiments, during assembly, P1, a 95-kDa capsid polyprotein precursoris cleaved by the viral 3C protease to ultimately yield VP1, VP2, VP3and VP4.

FMDV P1 precursor polypeptide (or P1 precursor protein) is a polypeptidecomprised of the FMDV structural proteins and/or precursors, VP0, VP1,VP2, VP3, and VP4, as well as the 2A translational interrupter. The FMDVP1 precursor is around 85 kDa in molecular weight. The P1 precursor isprocessed by the FMDV 3C protease into structural proteins forming VLPsand the FMDV capsid.

The FMDV VP0 protein is a precursor peptide comprised of the FMDV VP2and VP4 structural proteins. The FMDV VP0 protein is also identified asthe FMDV 1AB protein and is around 33 kDa in molecular weight. It isproduced by the processing of the FMDV P1 precursor protein by the FMDV3C protease. The FMDV VP0 protein is important in the formation ofprotomers along with FMDV proteins VP3 and VP1. Five of these protomersassemble into a pentamer and twelve pentamers can assemble into a FMDVcapsid or VLP. Cleavage of VP0 into VP2 and VP4 occurs through anunknown mechanism.

The FMDV VP1 protein is a structural protein that comprises the FMDVcapsid and/or FMDV VLP. The FMDV VP1 protein is also identified as theFMDV 1D protein and is around 24 kDa in molecular weight. The FMDV VP1protein contains a mobile loop structure, identified as the G-H loop,which emerges from the surface of the FMDV capsid and/or VLP. The FMDVVP1 protein can form a protomer along with VP0 and VP3. Five of theseprotomers assemble into a pentamer and twelve pentamers can assembleinto a FMDV capsid or VLP.

The FMDV VP2 protein is a structural protein that comprises the FMDVcapsid and/or FMDV VLP. The FMDV VP2 protein is also identified as theFMDV 1B protein and is around 24 kDa in molecular weight. The FMDV VP2protein, along with the FMDV VP4 protein, is part of the FMDV VP0protein until the formation of FMDV capsids and/or VLPs at which pointthe VP0 protein is processed into VP2 and VP4.

The FMDV VP3 protein is a structural protein that comprises the FMDVcapsid and/or FMDV VLP. The FMDV VP3 protein is also identified as theFMDV 1C protein and is around 24 kDa in molecular weight. The FMDV VP3protein can form a protomer along with VP0 and VP1. Five of theseprotomers assemble into a pentamer and twelve pentamers can assembleinto a FMDV capsid or VLP.

The FMDV VP4 protein is the smallest of the FMDV structural proteins andis part of the FMDV capsid and/or FMDV VLP. The FMDV VP4 protein is alsoidentified as the FMDV 1D protein and is around 9 kDa in molecularweight. The FMDV VP4 protein, along with the FMDV VP2 protein, is partof the FMDV VP0 protein until the formation of FMDV capsids and/or VLPsat which point the VP0 protein is processed into VP2 and VP4. Unlikeother FMDV proteins that comprise the capsid and/or VLP the VP4 proteinis entirely located inside the capsid and/or VLP structure.

“Virus-like particles” or “VLPs” resemble viruses, but arenon-infectious because they do not contain any viral genetic material.The expression of viral structural proteins, such as envelope or capsid,can result in the self-assembly of VLPs that can stimulate an immuneresponse in a mammalian organism. In other words, VLPs are often emptyviral envelopes or empty viral capsids that are capable of stimulatingan immune response like a full virus. Methods and problems associatedwith the production of VLPs in alternative systems include thosedescribed by Lee, et al., J. Biomed. Sci. 16:69 (Aug. 11, 2009),Srinivas, et al., Biologicals 44:64-68 (2016), Mayr, et al., Vaccine 19:2152-2162 (2001) and Niborski, et al., Vaccine 24: 7204-7213 (2006) thatare each incorporated by reference.

According to various embodiments of the method, the lysis buffercontains Tris-HCl, NaCl, and Triton X-100. Tris is short fortris(hydroxymethyl)aminomethane. The useful buffer range for tris (7-9)coincides with the physiological pH typical of most living organisms.This, and its low cost, make tris one of the most commonly used buffers.NaCl to help keep proteins soluble and to mimic physiologicalconditions. NaCl can also be used to lyse cells through osmosis. TritonX-100 is a nonionic surfactant that has a hydrophilic polyethylene oxidechain (on average it has 9.5 ethylene oxide units) and an aromatichydrocarbon lipophilic or hydrophobic group Triton X-100 is a commonlyused detergent in laboratories. Triton X-100 is widely used to lysecells to extract protein or organelles, or to permeabilize the membranesof living cells.

In some embodiments, the lysis buffer does not containethylenediaminetetraacetic acid (EDTA). In some embodiments, the lysisbuffer further contains MgCl₂. In some embodiments, the lysis buffercontains 10-20 mM Tris-HCl, 150-200 mM NaCl, and 1% Triton X-100 (w/v).

In some embodiments, the lysis buffer contains 10-20 mM Tris-HCl,150-200 mM NaCl, 3 mM MgCl₂, and 1% Triton X-100 (w/v). In anembodiment, the lysis buffer contains 20 mM Tris-HCl, 200 mM NaCl, 3 mMMgCl₂, and 1% Triton X-100 (w/v) and does not contain EDTA.

In embodiments of the method, cells containing VLPs are contacted withthe lysis buffer and the cells allowed to lyse. A solution of the lysedcells is then centrifuged to pellet the cellular components, while theVLPs remain in the supernatant. The supernatant containing the VLPs isthen removed.

According to various embodiments, the method of purifying VLPs furtherincludes centrifuging the supernatant through a gradient, such as acesium chloride gradient, a sucrose gradient, or both.

According to various embodiments, the cells containing the VLPs areeukaryotic cells. In some embodiments, the cells are mammalian cells. Insome embodiments, the cells are insect cells. In further embodiments,the cells are CHO-K7 or HEK293-T cells.

According to various embodiments of the method, the purified VLPs areimmunogenic and capable of inducing an immune response when an effectiveamount is inoculated in animals. In embodiments, the purified VLPs arecapable of eliciting protection from foot-and-mouth disease (FMD) in theinoculated animal.

In a second aspect, the present disclosure provides a method ofinoculating an animal to induce an immune response against FMDV. Inembodiments, the method induces an immune response that protects theanimal from FMDV.

The method of inoculating includes (i) providing FMDV VLPs that havebeen purified by a method that includes contacting cells containing FMDVVLPs with a lysis buffer that contains Tris-HCl, NaCl, and Triton X-100,centrifuging a solution of the lysed cells, and removing the supernatantcontaining the purified FMDV VLPs; and (ii) inoculating the animal withan effective amount of the purified FMDV VLPs.

According to various embodiments, the lysis buffer does not containEDTA. In some embodiments, the lysis buffer further contains MgCl₂. Insome embodiments, the lysis buffer contains 10-20 mM Tris-HCl, 150-200mM NaCl, and 1% Triton X-100 (w/v). In some embodiments, the lysisbuffer contains 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl₂, and 1%Triton X-100 (w/v). In an embodiment, the lysis buffer contains 20 mMTris-HCl, 200 mM NaCl, 3 mM MgCl₂, and 1% Triton X-100 (w/v) and doesnot contain EDTA.

According to various embodiments, the FMDV VLPs are from one or more ofFMDV serotypes A, O, C, Asia, SAT1, SAT2, and SAT 3. In someembodiments, the VLPs are from one or more of FMDV strains A24 Cruzeiro,A2006 Turkey, O1 Campos, O1 Manisa, O Pan Asia II, O Hong Kong, C3Indaial, Asia 1 Shamir, SAT1 KNP91, SAT3 ZIM91, and SAT2 Egypt 2010.According to various embodiments, the animal is a mammal. In someembodiments, the animal is a goat, a sheep, a pig, or a cow.

According to various embodiments of the method of inoculating an animal,the immune response to FMDV is effective to provide protection againstchallenge with FMDV. In some embodiments, the effective amount of theFMDV VLPs is an amount effective to produce an immune response thatprotects the animal against challenge with FMDV. In some embodiments,the animal is inoculated with a vaccine containing an effective amountof the FMDV VLPs.

A vaccine in accordance with the present disclosure is a biologicalcomposition that provides or improves immunity to an organism to aparticular disease. A vaccine may contain an agent, such as a killed,inactivated, weakened or attenuated form of the disease-causingmicroorganism (e.g., virus, bacteria, fungi, algae), its toxins, surfaceproteins or recombinant nucleic acid such as DNA, compositions orparticles that resemble the pathogenic microorganism (e.g., virus-likeparticles) or combinations thereof. The agent functions as an antigenand is administered to an organism to stimulate the body's immune systemto produce an immune response, that may include recognizing the agent asforeign, destroying the agent (e.g., with antibodies produced that arespecific to the agent/antigen), and remembering the agent, so the immunesystem can more easily recognize and destroy any of these microorganismsthat it later encounters, for example, an infection.

Virus-like particles, or VLPs, can be used in accordance withembodiments of the present disclosure. VLPs are recombinant particleswith viral matrix or structural proteins such as capsids that resembleviruses, but are non-infectious and unable to propagate as they,respectively, do not contain any viral genetic material. VLPs can beutilized as vaccine antigens as they mimic the native virions, and canbe produced in vitro in a variety of cell culture systems includingmammalian cell lines, insect cell lines, yeast and plant cells or invivo. In embodiments, FMDV VLPs consist essentially of assembledstructural proteins or assembled capsid proteins (e.g., VP1, VP2, VP3and VP4).

In a third aspect, the present disclosure provides a compositioncontaining VLPs purified by a method that includes contacting cellscontaining VLPs with a lysis buffer that contains Tris-HCl, NaCl, andTriton X-100; centrifuging a solution of the lysed cells; and removingthe supernatant containing the purified VLPs. According to variousembodiments, the VLPs are FMDV VLPs.

In some embodiments, the lysis buffer consists of 10-20 mM Tris-HCl,150-200 mM NaCl, and 1% Triton X-100 (w/v). In some embodiments, thelysis buffer consists of 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl₂,and 1% Triton X-100 (w/v).

In various embodiments, the FMDV VLPs are from one or more of FMDVserotypes A, O, C, Asia, SAT1, SAT2, and SAT 3. In some embodiments, theVLPs are from one or more of FMDV strains A24 Cruzeiro, A2006 Turkey, O1Campos, O1 Manisa, O Pan Asia II, O Hong Kong, C3 Indaial, Asia 1Shamir, SAT1 KNP91, SAT3 ZIM91, and SAT2 Egypt 2010.

According to various embodiments, the composition is a vaccine for FMDcontaining an effective amount of FMDV VLPs. In some embodiments, thevaccine further comprises an adjuvant.

EXPERIMENTAL DATA Example 1 Buffer Comparisons

An initial selection of six lysis buffers was evaluated in a method forpreparing VLP's. The buffers included four lysis buffers, LB1-LB4, thatwere selected from an internet and literature search of buffers usedwith cytosolic viruses, specifically Herpesviruses. Two commerciallyproduced buffers, LB5 and LB6, were also included in the testing toserve as benchmarks by which other lysis buffers could be compared. LB5is M-PER™ buffer available from Thermo Scientific and contains aproprietary detergent in 25 mM bicine buffer (pH 7.6) (M-PER MammalianProtein Extraction Reagent User Manual, Thermo Scientific). LB6 isXTRACTOR™ buffer available from Clontech Laboratories and is based on amild non-ionic detergent (xTractor Buffer & xTractor Buffer Kit UserManual, Clontech Laboratories).

The components of the lysis buffers are shown in Table 1. The exactcomponents of the commercial buffers LB5 and LB6 are not publicly known.

TABLE 1 ID Source Components LB1 Internet Search 10 mM Tris-HCl, 200 mMNaCl, 10 mM EDTA, 1% Triton X-100 LB2 Internet Search 50 mM Tris-HCl,150 mM NaCl, 10 mM EDTA, 1% Triton X-100 LB3 Internet Search 20 mMTris-HCl, 150 mM NaCl, 3 mM MgCl2 LB4 Internet Search 20 mM Tris-HCl,150 mM NaCl, 3 mM MgCl2, 10 mM EDTA LB5 Commercial, MPER buffer LB6Commercial, Xtractor buffer LB7 Derived from LB 3 20 mM Tris-HCl, 150 mMNaCl, 3 mM MgCl2, 1% Triton X-100 LB8 Derived from LB 3 20 mM Tris-HCl,200 mM NaCl, 3 mM MgCl2 LB9 Derived from LB 3 20 mM Tris-HCl, 200 mMNaCl, 3 mM MgCl2, 1% Triton X-100 LB10 Derived from LB1 10 mM Tris-HCl,200 mM NaCl, 1% Triton X-100

The lysis buffers were evaluated by western blot with the extractedsupermatant and the F14 antibody, which detects FMDV VP0 and VP2.Transfected mammalian cell cultures were used to produce VLPs in asimilar means as in U.S. Pat. No. 10,385,319, the contents of which isincorporated by reference in its entirety, with some modifications. Themodifications include the utilization of polyethylenimine, MW 25000, asa transfection reagent instead of LIPOFECTAMINE™ 2000. Transfection ofcell cultures utilized the pJJP O1M-3C(L127P)-GLuc plasmid, FIG. 1A. Inaddition to the FMDV P1-2A and 3C protease coding sequences this plasmidcontains the sequence to Gaussia luciferase, which can be measured usinga luciferase assay to quickly quantify differences in proteinconcentrations. The results of lysis of transfected cells with LB1, LB2,LB3, LB4, LB5, and LB6 are shown in FIG. 1B. Of tested, LB3 performedthe best of the non-commercial buffers. Buffers containing EDTAunderperformed (LB1, LB2, and LB4).

Based on initial results of underperformance of EDTA containing buffers,four additional lysis buffers were formulated. These buffers areidentified as LB7 through LB10 and were compared to LB3 to determine iffurther enhancement in VLP extraction could be obtained by the additionof Triton X-100, addition of MgCl₂, and/or an increase in the amount ofNaCl (Table 1). Cell supernatants were evaluated for their luciferaseactivity, FIG. 2A, and by Western blot with the F14 antibody, FIG. 2B.

As shown by the results in FIGS. 2A and 2B, lysis buffers LB7, LB9, andLB10 showed improvement over LB3, with LB9 being the top performer.

Example 2 Luciferase Assay

Luciferase assay was performed by removal of cells from flasks andcentrifugation at 500×g for 10 minutes. Media was removed and cellswashed twice with 5 mL of DPBS to ensure the removal of any residualmedia. Cell pellets were resuspended in 2 mL of each tested lysis bufferseparately and allowed to incubate on a rocker for 10 minutes to inducecell lysis. Luciferase assay was performed using LUMITRAC white 96 wellplates (Greiner) in a 96-well BioSystems Veritas luminometer (TurnerBiosystems) with a mixture of 10 ul of cell lysis mixed with 90 ul ofddH2O in triplicate wells for samples. An injection of 100 ul of 50ug/mL of water-soluble coelentrazine (NanoLight Technologies) with anintegration time of 0.5 seconds before and after injection of substrate.Data was recorded in the form of relative luciferase units (RLUs) perhalf second.

Example 3 Additional Purification—CsCl₂ Gradient

Subsequent experiments used both LB3 and LB9 to extract VLPs of FMDVstrain O1 Manisa from CHO-K7 and HEK293-T cells for comparison by cesiumchloride gradient. Cell lysis products were layered on 2 mL cesiumchloride 2-step discontinuous gradients (1.42 g/cm³/1.38 g/cm3) preparedin TEN buffer (0.05M Tris, 0.001 M EDTA, 0.15 M NaCl, pH 7.4). Sampleswere centrifuged in a SW40Ti rotor at 217,485×g for 18 hours using anOptima L-80 XP ultracentrifuge (Beckman Coulter). Individual visiblebands were collected and dialyzed against PBS at 4° C. using 10K MWCOSlide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific).Post-dialysis samples were run on western blots and analyzed withmonoclonal antibody F1412SA.

Cesium chloride gradients are shown in FIG. 3. The material bandingbetween the black marks represents VLPs at the appropriate size rangefor FMDV VLPs. In this test, LB9 continued to outperform LB3.

The VLP Bands were extracted and dialyzed into phosphate bufferedsaline, and then used for Western blot analysis with F14 antibody. Theresults are shown in FIGS. 4A and 4B.

FIG. 4A is a cesium chloride gradient of VLPs extracted from HEK293-Tcells using lysis buffer LB9. In FIG. 4B, Upper (U) and Lower (L) bandswere extracted and run on the Western blot, along with an aliquot of VLPsupernatant that was not applied to a cesium chloride gradient(Pre-CsCl₂), using the F14 antibody for detection of the FMDV VP0 andVP2 proteins.

Lysis buffer LB9 was tested for the ability to extract VLPs from severaldifferent FMDV strains representative of four different serotypes, A,Asia, O, and SAT2. FIG. 5 shows cesium chloride gradients of the VLPsextracted from HEK293-T cells using LB9. The hazy material bandingbetween the black marks on the tubes are VLPs, that are at theappropriate size range for FMDV VLPs. By this analysis, LB9 is capableof purifying VLPS from each strain and serotype tested.

Lysis buffer LB9 was tested for the ability to extract antigen fromseveral different FMDV strains/serotypes. Results of the Western blotanalyses in FIG. 6 show that LB9 is capable of extracting antigen foreach strain and serotype tested.

Example 4 Swine Challenge Study of Vaccines

Animals were vaccinated in a prime/boost format with different dosagesof extracted VLPs. Boost vaccination occurred 14 days after the primevaccination and 7 days prior to challenge. For contact challenge studiesin swine, five naïve unvaccinated pigs were infected via intradermalheel bulb (IDHB) inoculation with FMDV O1 Manisa and co-mingled withvaccinated animals, as well as naïve unvaccinated sentinel animals.Animals were inspected for the presence of vesicular lesions at 1, 3, 5,7, 10, and 14 days post challenge. Animals were considered protectedfrom clinical disease if no lesions were observed for the duration ofthe study. For all challenge studies, 100% of sentinel animalsdemonstrated clinical FMDV lesions confirming disease spread.

Vaccines using VLPs extracted with LB9 and VLPs extracted by a previousmethod (Qproteome Cell Compartment Kit, Qiagen) were compared in a swinechallenge study with FMDV O1 Manisa. The results presented in Table 2show that LB9 extracted VLPs protected five of five swine from O1 Manisachallenge, while the Qproteome lysis buffer protected four of five fromchallenge. This study demonstrates that utilization of LB9 to extractantigen does not render the vaccine non-functional. While the samplenumber in this study is limited, the LB9 extracted VLPs may produce asuperior result than the previous Qproteome method (5/5 compared to4/5).

TABLE 2 Animal No./ VNT Gender 0 dpcc 3 dpcc 6 dpcc 8 dpcc 10 dpcc 14dpcc Conclusion Qproteome 51824 M 2.4 NEG NEG NEG NEG POS(2) UnprotectedLysis Buffer 51830 F 1.8 NEG NEG NEG NEG NEG Protected extracted 51837 F2.1 NEG NEG NEG NEG NEG Protected VLPs 51839 M 0.9 NEG NEG NEG NEG NEGProtected 51840 M 1.5 NEG NEG NEG NEG NEG Protected Lysis Buffer 9 51826M 2.1 NEG NEG NEG NEG NEG Protected Extracted 51829 M 0.9 NEG NEG NEGNEG NEG Protected VLPs 51833 F 1.2 NEG NEG NEG NEG NEG Protected 51836 F2.1 NEG NEG NEG NEG NEG Protected 51841 M 1.2 NEG NEG NEG NEG NEGProtected

Usage of LB9 results in a dramatic reduction in potential costs forproducing VLPs as LB9 costs roughly $0.005 per dose in reagents, while acommonly used commercial lysis buffer costs about $31.00 per dose (e.g.,Qproteome Cell Compartment Kit, Qiagen).

Animals were vaccinated in a prime/boost format with different dosagesof either QLB or LB9 derived VLPs are shown in Table 3.

TABLE 3 Dosage % Method n (ml) Protected LB9 - VLP 5 0.825 100%  LB9 -VLP 5 0.6 100%  QLB - VLP 5 1.4 60% QLB - VLP 5 0.75 80% QLB - VLP 50.35 80% QLB - VLP 5 0.0875 40%

Example 5 Determination of Virus Neutralizing Titers (VNTs)

Virus neutralizing antibody titers against FMDV serotype O1 Manisa wasdetermined by VNT on BHK-21 cells as per World Organisation for AnimalHealth (OIE) protocols. Neutralization titers are expressed as the log₁₀of the reciprocal of the highest serum dilution resulting in 50%neutralization of the cytopathic effect (Spearman-Kärber method).

VNT by Method

In FIG. 7, the highest observed VNTs for each pig during the course ofthe experiment was sorted in relation to VLPs extracted with either QLB(black) or LB9 (gray). This data was sorted into one of three groups,VNTs less than 1.0, VNTs between 1.0 and 2.0, and VNTs above 2.0. Foranimals vaccinated with LB9, 70% had VNTs above 2.0 at some point duringthe experiment compared to only 20% of animals vaccinated with QLB. Amajority of animals, over 70%, vaccinated with QLB presented with VNTsin the range of 1.0 and 2.0.

VNT by Group

The VNTs at the time of challenge of all animals within a treatmentgroup were averaged together and are presented in FIG. 8. Changes indosage did not have an effect on the VNTs resulting from VLPs extractedwith QLB, while it did have an effect on the VNTs from VLPs extractedwith LB9. This suggests that VLPs extracted with QLB and LB9 arestimulating the immune system in different ways.

The foregoing disclosure provides examples of specific embodiments. Aswill be understood by those skilled in the art, the approaches, methods,techniques, materials, devices, and so forth disclosed herein may beembodied in additional embodiments as understood by those of skill inthe art, it is the intention of this application to encompass andinclude such variations. Accordingly, this disclosure is illustrativeand should not be taken as limiting the scope of the following claims.

1. A method of producing purified VLPs, comprising: a) contacting cellscontaining VLPs with a lysis buffer and allowing the cells to lyse, thelysis buffer comprising: 10-20 mM Tris-HCl, 150-200 mM NaCl, 3 mM MgCl₂,and 1% Triton X-100, wherein the lysis buffer does not contain EDTA; b)centrifuging a solution of step (a); and c) removing a supernatant fromthe solution of step (b), the supernatant containing the purified VLPs.2. The method of claim 1, further comprising centrifuging thesupernatant through a cesium chloride and/or sucrose gradient.
 3. Themethod of claim 1, wherein the cells are eukaryotic cells.
 4. The methodof claim 1, wherein the cells are mammalian cells.
 5. The method ofclaim 1, wherein the cells are insect cells.
 6. The method of claim 1,wherein the cells are CHO-K7 or HEK293-T cells.
 7. The method of claim1, wherein the VLPs is selected from Swine Vesicular Disease Virus,Senecavirus A, Porcine Teschovirus, Bovine rhinitis A virus, Bovinerhinitis B virus, Equine rhinitis A virus, or Foot-and-mouth diseasevirus (FMDV).
 8. A method of inoculating an animal to induce an immuneresponse to viral disease, comprising: a) providing purified VLPsproduced according to the method of claim 1; and b) inoculating theanimal with an effective amount of the VLPs.
 9. The method of claim 8,wherein the viral disease is selected from Swine Vesicular DiseaseVirus, Senecavirus A, Porcine Teschovirus, Bovine rhinitis A virus,Bovine rhinitis B virus, Equine rhinitis A virus, or Foot-and-mouthdisease virus (FMDV)
 10. The method of claim 8, wherein the animal is amammal.
 11. The method of claim 8, wherein the animal is a pig, cow,goat, horse or sheep.
 12. The method of claim 11, wherein the animal isa cow.
 13. The method of claim 11, wherein the animal is a pig.
 14. Themethod of claim 8, wherein the animal is inoculated with a vaccinecontaining the effective amount of the VLPs.
 15. The method of claim 8,wherein the immune response is effective to provide protection againstviral challenge.
 16. The method of claim 8, wherein the effective amountof the VLPs is an amount effective to produce an immune response thatprotects the animal against challenge with live virus.
 17. A compositioncomprising purified VLPs produced by the method according to claim 1.18. The composition of claim 17, wherein the composition is a vaccinefor comprising an effective amount of the VLPs.
 19. The method of claim18, wherein the virus is selected from Swine Vesicular Disease Virus,Senecavirus A, Porcine Teschovirus, Bovine rhinitis A virus, Bovinerhinitis B virus, Equine rhinitis A virus, or Foot-and-mouth diseasevirus (FMDV)
 20. The composition of claim 19, further comprising anadjuvant.